In view of the progress in biotechnology and cancer therapies, it is important to elucidate mechanisms involved in cell division control and immortalization. There is a limitation to the number of cell divisions in the cases of normal cells. Ultimately, cells reach a metabolically active state that is referred to as replicative senescence, or, in other words, permanent growth arrest (Non-Patent Document 33). However, when genetic or epigenetic changes are induced by certain mechanisms, it becomes possible for cells to continue to be divided permanently in culture while escaping the limitations of division capacity. This can be referred to as a state of cell “immortalization.” In a very few cases, cells may spontaneously be immortalized. Control of the initiation and the termination of cell division at the molecular level have not so far been completely elucidated. For instance, when a viral oncogene is expressed in cells, the cell lifespan is extended. Then, such cells enter a stage that is referred to as “crisis.” Very rarely (at an incidence of 10−9 to 10−6), few cells can escape from such “crisis” and become immortalized. Molecular basis of the phenomena such as, cell immortalization, malignant transformation, and tumor growth or development have not been elucidated. Although studies on the involvement of intracellular factors such as telomerase in the phenomena have been gaining attention, there are many studies suggesting that immortalization may take place by telomerase-independent telomere maintenance. These studies have suggested the existence of a certain senescence mechanism that is unrelated to telomeres, and the role of genes or routes that are independent from telomerase activity (Non-Patent Documents 34-38).
Mortalin is a protein involved in a variety of intracellular functions such as intracellular signal transduction, cell differentiation, and cell division control. A gene encoding mortalin was first isolated as an hsp 70 family protein present in the cytoplasmic fraction of a normal mouse fibroblast (Non-Patent Document 1). Subsequently, it has been revealed that such protein does not exist in the cytoplasmic fraction of an immortalized fibroblast. An antibody against a full-length mortalin protein that had been isolated from a normal fibroblast was produced (Non-Patent Document 1), and the resulting antibody was used for immunofluorescent staining. As a result, the staining was detected in the cytoplasm in normal cells. On the other hand, in the immortalized cells the staining was detected in the perinuclear region (Non-Patent Document 2).
Further, based on immunocloning of cDNA from a mouse-immortalized cell and comparison of that with the sequence isolated from a normal cell, it was revealed that two types of mortalin genes (mot-1 and mot-2) encoding different proteins exist. The proteins differ from each other only in 2 amino acid residues in the carboxyl terminal (Non-Patent Document 3). mot-1 (mortalin-1) exists in normal and mot-2 (mortalin 2) exists in immortalized cells.
Studies using NIH 3T3 cells have revealed that cDNAs of these two genes have contrasting biological activities. It has been revealed that the expression of mot-1 (mortalin 1) results in the generation of a cell-senescence-like phenotype. On the other hand, it has been revealed that, based on nude mouse assay, the overexpression of mot-2 (mortalin 2) causes malignant transformation (Non-Patent Document 4).
In the beginning of the mortalin study, it was not revealed whether mot-1 and mot-2 would be two different genes or alleles of each other (Non-Patent Documents 5 and 6). The ultimate answer was obtained based on mouse family study. Since it was found that two gene loci had been separated in mice in two generations, it became clear that mot-1 and mot-2 are alleles in the same loci in a mouse (Non-Patent Document 7).
Mortalin 2 was also identified as PBP74 (Non-Patent Document 8) mtHSP70 (Non-Patent Document 9), and GRP75 (Non-Patent Document 10). It has been remarked that mortalin 2 is involved in various functions related to stress response (Non-Patent Documents 10-15), intracellular transport (Non-Patent Document 11), antigen processing (Non-Patent Document 8), cell growth control (Non-Patent Documents 3, 4, and 12), control of in vivo nephrotoxicity (Non-Patent Documents 13 and 14), differentiation (Non-Patent Document 15) tumorigenesis (Non-Patent Documents 4 and 16), and so on.
In particular, mortalin 2 has been confirmed to bind to p53, a tumor suppressor protein, such that mortalin 2 inactivates the transcriptional activity of p53 (Non-Patent Document 17). Such p53 inactivation has been considered to be one of the causes of NIH 3T3 malignant cell transformation (Non-Patent Document 4) and lifespan prolongation of normal human fibroblast (Non-Patent Document 18). In addition, mortalin 2 has been found to cooperate with telomerase to immortalize a human foreskin fibroblast (Non-Patent Document 19).
In contrast with murine cells, human cells contain only one type of mortalin. Human mortalin has activity similar to that of murine mortalin 2 (mot-2), and thus it was referred to as hmot-2 (Non-Patent Document 4). In both mice and human cells, mortalin exists at multiple intracellular sites. Thus, it has been suggested that there are at least two types of mechanisms that are responsible for localized intracellular distribution of mortalin proteins (Non-Patent Document 20). The first mechanism is established based on the existence of different types of cDNAs, involving two alleles that have been found in mice (mot-1 and mot-2).
The second mechanism may involve unknown protein modifiers or cell factors to be found in both mice and humans.
In both the cases of humans and mice, upon detection of mortalin by staining with an antibody, the distribution of mortalin was confirmed throughout the cytoplasm in the case of normal cells, and in the cases of immortalized and tumor cells, it was localized to the perinuclear region. A human cell derived from an in vitro mutated tumor exhibits a mortalin staining pattern in which the distribution is not observed throughout the cytoplasm. However, a normal cell exhibits a staining pattern in which the distribution is observed throughout the cytoplasm (Non-Patent Document 21). When cell senescence was induced by introducing chromosome 7 into an SUSM1 cell, the mortalin staining pattern changed from the non-pancytoplasmic to the pancytoplasmic (Non-Patent Document 22). Also, in the case of induction of cell senescence with the use of 5-bromodeoxyuridine, similar changes in mortalin staining patterns have been observed (Non-Patent Documents 12 and 23). When human mutated cells experienced growth arrest by rhodacyanin dye treatment, changes in mortalin staining patterns were also observed (Non-Patent Document 24). Based on these studies, it has been shown that mortalin intracellular distribution is related to a phenotype for cell division.
Also, there are studies suggesting correlation between the mortalin expression level and the muscle or mitochondrial activity and differentiation (Non-Patent Documents 25 and 26). For instance, human mutated cells or tumor cell lines exhibit upregulation of mortalin expression (4). At the same time, mortalin expression level decreases during induction of differentiation in HL-60 promyelocytic leukocytes, (15). On the other hand, in mortalin-overexpressing cells, the level of differentiation induction was significantly reduced (Non-Patent Document 15).
Ssc1p is a homolog of mortalin in yeasts. Ssc1p is essential for cell viability (Non-Patent Document 27). In particular, Ssc1p has a function that is indispensable for mitochondrial transport (Non-Patent Document 28). Ssc1p is an essential constituent of a mitochondria transport apparatus, which binds to Tim-44, which is an inner mitochondrial membrane anchor (Non-Patent Documents 29 and 30). Mutations in Tim-44 that results in insufficient recruitment of mtsp70/Ssc1 are lethal to yeast cells (Saccharomyces cerevisiae) (Non-Patent Document 28). Based on studies of yeasts, mortalin has been estimated to have at least 3 types of activities. The activities include (i) unfolding of an extramitochondrial protein, (ii) one-way mitochondrial transmembrane transportation initiated by membrane potentials (M, Δ, and Ψ), and (iii) completion of transfer through action as an ATP-driving motor. Also, mortalin is necessary for intramitochondrial degradation of a misfolded peptide with m-AAA and PIM1 proteases. In addition, it has been suggested that mortalin cooperates with mtHSP60 and CPN10 chaperones intramitochondrially, so as to fold a transferred protein such that the protein is formed into a functionally useful form, and that mortalin is involved in unknown functions of mtHSP60 in the extramitochondrial environment (Non-Patent Documents 31 and 32). Based on these reports, in addition to inactivation of tumor suppressor p53, functions of mortalin serving as a mitochondria transport apparatus and as chaperonine are expected to contribute to cell division phenotypes. Mortalin is considered to be a protein that is responsible for a variety of functions that control cell division at different intracellular sites.
A novel anticancer agent has been awaited, such agent should target the molecules that are characteristic of cancer cells and are involved in cancer cell division, immortalization, and metastasis. Such agents should have little adverse effects on normal cells. In addition, the development of target therapies has also been awaited, whereby adverse effects caused by destruction of normal cells can be avoided by allowing drugs such as anticancer agents, which have strong effects of incidentally killing normal cells, to have the property of being delivered to cancer cells in lesions so as to attack such cancer cells.
Antibody medicines are promising as new anticancer agents that are expected to achieve the above objectives. With the use of antibodies against antigen proteins of cancer cells to be eliminated it is possible to attack the cancer cells exclusively in affected parts. In addition, it is also possible to use the antibody medicines for target therapies for delivering drugs against target antigens. It is expected that such target therapies exert high levels of therapeutic effects.    Patent Document 1: JP Patent Publication (Kokai) No. 2001-354564 A    Patent Document 2: JP Patent Application No. 11-272778    Patent Document 3: JP Patent Application No. 11-357545    Non-Patent Document 1: Wadhwa, R., Kaul, S. C., Ikawa, Y., and Sugimoto, Y. (1993) J Biol Chem 268, 6615-6621    Non-Patent Document 2: Wadhwa, R., Kaul, S. C., Mitsui, Y., and Sugimoto, Y. (1993) Exp Cell Res 207, 442-448    Non-Patent Document 3: Wadhwa, R., Kaul, S. C., Sugimoto, Y., and Mitsui, Y. 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